Enone reductase gene and microbial production of levodione

ABSTRACT

Disclosed is an isolated DNA comprising a nucleotide sequence coding for an enzyme having enone reductase activity wherein the enzyme is characterized by the following physico-chemical properties: (a) molecular mass: 61,300±5,000 Da (estimated using gel filtration, consisting of one subunit); (b) co-factor: NADPH and NADH; (c) substrate specificity: active on α,β-unsaturated ketons; (d) optimum temperature: 55-60° C. at pH 7.4; and (e) optimum pH: pH 4.5-8.5.

This application is the National Stage of International Application No.PCT/EP2003/010473, filed Sep. 19, 2003.

The present invention relates to a DNA encoding an enone reductase, anexpression vector comprising the DNA, a microorganism into which the DNAhas been introduced, and a method for producing(6R)-2,2,6-trimethyl-1,4-cyclohexanedione (hereinafter referred to aslevodione) from 2,6,6-trimethly-2-cyclohexene-1,4-dione (hereinafterreferred to as ketoisophorone) using the microorganism.

Levodione is a useful intermediate in the synthesis of optically activecarotenoids such as zeaxanthin. A microbiological process of producinglevodione from ketoisophorone is known (U.S. Pat. No. 4,156,100). Enonereductase that acts on ketoisophorone to produce levodione, which wasisolated from Candida kefyr was described in (European PatentApplication No. 02003967.3 filed on Feb. 22, 2002). This enzyme ischaracterized by the following physico-chemical properties:

-   -   (a) molecular mass: 61,300±5,000 Da        -   (Estimated using gel filtration. Consisting of one subunit.)    -   (b) Co-factor: NADPH and NADH    -   (c) Substrate specificity: active on α,β-unsaturated ketons    -   (d) Optimum temperature: 55-60° C. at pH 7.4    -   (e) Optimum pH: pH 4.5-8.5

As used herein, the term “enone reductase” encompasses proteinscatalyzing the enzymatic reduction of carbonyl activated double bondsaccording to the Enzyme Nomenclature provided by the NomenclatureCommittee of the International Union of Biochemistry and MolecularBiology (NC-IUBMB). It also relates to proteins having the abovementioned activities of an enone reductase, which proteins preferablycatalyse the conversion of ketoisophorone into levodione. The gene foran enone reductase involved in the biosynthesis of levodione would bevery useful for improvement of levodione productivity by amicroorganism.

The present invention provides to an isolated DNA sequence encodingenone reductase.

The isolated DNA sequence may be more specifically characterized in that(a) it codes for the enzyme having the amino acid sequence described inSEQ ID NO:2, or (b) it codes for a variant of the enzyme selected from(i) an allelic variant, and (ii) an enzyme having one or more amino acidaddition, insertion, deletion and/or substitution and having the statedenzyme activity.

More particularly, the present invention provides an isolated DNAsequence derived from a gene of Candida kefyr (Candida macedoniensis)IFO 0960 and is selected from (i) the DNA sequence represented in SEQ IDNO:1, (ii) an isocoding or an allelic variant of the DNA sequencerepresented in SEQ ID NO:1, (iii) a derivative of the DNA sequencerepresented in SEQ ID NO:1, with addition, insertion, deletion and/orsubstitution of one or more nucleotide(s) and coding for a polypeptidehaving the enzyme activity, (iv) the DNA sequence which hybridizes tothe complement of the nucleotide sequence of (i) or (ii) under stringenthybridizing conditions and coding for a polypeptide having the enzymeactivity, and (v) the DNA sequence which is at least 80% identical tothe nucleotide sequence of (i) and coding for a polypeptide having theenzyme activity.

The strain Candida kefyr (Candida macedoniensis) IFO 0960 is publiclyavailable from the Institute for Fermentation Osaka (IFO), 17-85Juso-honmachi 2-chome, Yodogawa-ku, Osaka, 532-8686, Japan.

Instructions for identifying DNA sequences by means of hybridization arewell-known to a person skilled in the art. The hybridization may takeplace under stringent conditions wherein only hybrids in which the probeand target sequence, i.e. the polynucleotides treated with the probe andare at least 70% identical, are formed. It is known that the stringencyof the hybridization, including the washing steps, is influenced ordetermined by varying the buffer composition, the temperature and thesalt concentration. The hybridization reaction is preferably carried outunder a relatively low stringency compared with the washing steps.

A 5×SSC buffer at a temperature of approx. 50-68° C., for example, canbe employed for the hybridization reaction. Probes can also hybridizehere with polynucleotides that are less than 70% identical to thesequence of the probe. Such hybrids are less stable and are removed bywashing under stringent conditions. This can be achieved, for example,by lowering the salt concentration to 2×SSC and subsequently 0.5×SSC ata temperature of approx. 50-68° C. being established. It is optionallypossible to lower the salt concentration to 0.1×SSC. Polynucleotidefragment, for example, at least 70% or at least 80%, or at least 90% to95% identical to the sequence of the probe employed can be isolated byincreasing the hybridization temperature stepwise in steps of approx.1-2° C.

“Stringent conditions” in the context of this invention meanhybridization in a buffer, for example, consisting of 5 ×SSC, 0.1% (w/v)N-lauroylsarcosine, 0.02%(w/v) SDS, 1% blocking reagent (RocheDiagnostics, Cat. No. 1096 176) at 50° C. overnight and two times ofwashing with 2×SSC, 0.1% (w/v) SDS for 5 min. at room temperature andfollowing two times of washing with 0.1×SSC, 0.1% (w/v) SDS for 15 min.at 68° C. in the washing step of hybridization.

The DNA sequence may be cloned from a strain of C. kefyr (C.macedoniensis) IFO 0960, or another or related organism and thus, forexample, may be an allelic or species variant of an enone reductaseencoding region of the DNA sequence. Also included within the scope ofthe present invention is a derivative of the DNA sequence with addition,insertion, deletion and/or substitution of different nucleotidesresulting in a polypeptide that encodes the same or a functionallyequivalent levodione reductase. The encoded protein may also containaddition, deletion, insertion and/or substitution of amino acidresidues, which produce a silent change and result in a functionallyequivalent enone reductase.

The DNA of the present invention also means a genomic DNA that containsregulatory sequences such as a promoter and a terminator, which areinvolved in the expression of the gene of interest, and also a cDNA thatcontains only open reading frame flanked between the short fragments inits 5′- and 3′-untranslated region.

The enone reductase gene, the recombinant expression vector, and therecombinant organisms utilized in the present invention may be obtainedby the following steps:

-   -   Isolating chromosomal DNA from a microorganism that can provide        enone reductase of the present invention and constructing the        gene library with the chromosomal DNA.    -   Cloning an enone reductase gene from the chromosomal DNA by        colony- or plaque-hybridization, PCR cloning, Southern-blot        hybridization and so on.    -   Determining nucleotide sequence of the enone reductase gene        obtained as above by usual methods and constructing recombinant        expression vectors which contain and express the enone reductase        gene efficiently.    -   Constructing recombinant organisms carrying the enone reductase        gene on recombinant expression vectors or on chromosomes by        transformation, transduction, transconjugation or        electroporation.

The techniques used to isolate or clone a DNA encoding enone reductaseof the present invention are known in the art and include isolation fromgenomic DNA. The cloning of the DNA sequence of the present inventionfrom such genomic DNA can be effected by using the degenerate polymerasechain reaction (hereinafter referred to as PCR).

On the basis of information on the partial amino acid sequenceoligonucleotides as primers for PCR may be synthesized. The primers usedfor cloning of the enone reductase gene by PCR may be based on the aminoacid sequence of the peptide fragments of the purified enone reductasefrom the genera including, but not to limited to, Candida andZygosaccharomyces, and in the most preferred embodiment, from C. kefyr(C. macedoniensis) IFO 0960. A DNA fragment (a partial DNA sequence) ofenone reductase is generated by PCR amplification with the primers andthe template of, e.g., C. kefyr chromosomal DNA. The amplified DNAfragment can be used as the probe to clone a genomic fragment coding forthe whole enone reductase. An entire gene containing its coding regionas well as its regulation region such as a promoter or terminator can becloned from a chromosome, for example, by inverse PCR method usingprimers based on part of sequence of the obtained DNA fragment after itwas sequenced, or screening of genomic library which is constructed inphage vector or plasmid vector in an appropriate host, by using apartial DNA fragment obtained by PCR as described above as a probe afterit was labeled.

Generally, E. coli as a host strain and E. coli vector, a phage vectorsuch as λ phage vector, a plasmid vector, or a yeast vector is oftenused in the construction of library and a following genetic manipulationsuch as sequencing, restriction digestion, ligation and so on. After theisolation of all necessary parts of the entire gene containing itscoding region as well as its regulation region, obtained fragments weresubcloned into an appropriate plasmid vector, which can be convenientlyused for sequencing and construction of the entire gene of the enonereductase. In this invention, the insert fragments were subdoned intopUC18 vector. Nucleotide sequence can be determined by a well-knownmethod such as dideoxy chain-termination method.

The isolated DNA sequence of the present invention may be used toidentify and clone DNA encoding a polypeptide having enone reductaseactivity from other strains of different genera or species according tomethods well known in the art.

The present invention also relates to a recombinant DNA, preferably avector and/or plasmid comprising a sequence coding for enone reductase.The recombinant DNA vector and/or plasmid may comprise the regulatoryregions such as promoters and terminators as well as open reading framesof a enone reductase gene. Methods which are well known to those skilledin the art may be used to construct expression vectors containing anucleotide sequence encoding enone reductase and appropriatetranscriptional and translational regulatory elements including allcomponents which are necessary or advantageous for expression of thecoding sequence of the nucleotide sequence. Specific initiation andtermination signals may also be used to achieve more efficienttranslation of sequences encoding enone reductase. An isolated DNAsequence encoding enone reductase may be manipulated in a variety ofways to provide for expression of the polypeptide. Manipulation of thenucleotide sequence encoding enone reductase prior to its insertion intoa vector may be desirable or necessary depending on the expressionvector. The techniques for modifying nucleotide sequences utilizingcloning methods are well known in the art. A variety of expressionvector/host systems may be utilized to contain and express sequencesencoding enone reductase.

The present invention also provides the use of the recombinant DNA totransform a host organism. A convenient form of the recombinant DNA maybe a vector. The host organism transformed with the recombinant DNA maybe useful in the production of a polypeptide of enone reductase and alsouseful in the improvement of the production process of levodione. Thus,the present invention also provides such a transformed host cell(recombinant microorganism) and a polypeptide encoded by the recombinantDNA.

The present invention also provides a process for the production of thepolypeptide encoded by the recombinant DNA, which comprises culturingthe transformed host cell under the conditions suitable for theexpression of the enzyme and recovery of the polypeptide from the cellculture. Cultivation of the recombinant microorganism can be carried outaerobically or anaerobically at pH values from 4.0 to 9.0, at atemperature in the range of from 10 to 60° C., for 15 minutes to 72hours, preferably, at pH values from 5.0 to 8.0, at a temperature in therange of from 20 to 40° C. for 30 minutes to 48 hours. The enonereductase produced by the recombinant cell may be secreted or containedintra-celluarly depending on the sequence and/or the vector used. Theenone reductase may then be isolated from the culture medium or therecombinant cell by conventional procedures.

The present invention further provides a process for the production oflevodione, which comprises contacting ketoisophorone with thepolypeptide enone reductase.

The enone reductase of the present invention catalyzes the reduction ofketoisophorone to levodione in the presence of a co-factor, NADH orNADPH, according to the following formula:Ketoisophorone+NADH (NADPH)

Levodione+NAD (NADP)

The reaction can be conducted in a solvent such as Tris-HCl buffer andphosphate buffer.

Preferable conditions for the reaction are pH values from 4.5 to 8.5,more preferably from 5.0 to 8.0, a temperature range of from 10 to 60°C., more preferably from 20 to 60° C., for a period of 5 minutes to 72hours, more preferably for 15 minutes to 48 hours.

The present invention also provides a method for the biologicalproduction of levodione, which comprises contacting ketoisophorone witha recombinant microorganism as described above, including cultivation ofthe recombinant microorganism in the presence of ketoisophorone as asubstrate, under conditions suitable for the production of levodione,and isolating the resulting levodione from the reaction mixture.

Either a growing or a resting cell culture or immobilized cells or acell-free extract, or the like, of the recombinant microorganism may beused for the production of levodione.

The growing cell culture can be obtained by culturing the recombinantmicroorganism in a nutrient medium containing saccharides such asglucose or sucrose, alcohols, such as ethanol or glycerol, fatty acids,such as oleic acid and stearic acid or esters thereof, or oils, such asrapeseed oil or soybean oil, as carbon sources; ammonium sulfate, sodiumnitrate, peptone, amino acids, corn steep liquor, bran, yeast extractand so on, as nitrogen sources; magnesium sulfate, sodium chloride,calcium carbonate, potassium monohydrogen phosphate, potassiumdihydrogen phosphate, and so on, as inorganic salt sources; and maltextract, meat extract, and so on, as other nutrient sources. Cultivationof the recombinant microorganism can be carried out aerobically oranaerobically at pH values from 4.0 to 9.0, at a temperature in therange of from 10 to 60° C. for 15 minutes to 72 hours, preferably, at pHvalues from 5.0 to 8.0, at a temperature in the range of from 20 to 40°C. for 30 minutes to 48 hours. Appropriate mixing of the culture duringthe cultivation will be preferable for the cell growth or the reaction.

Using the growing cell culture thus obtained, a resting cell culture orimmobilized cells or a cell-free extract may be prepared by any meansgenerally known in the art.

Preferable conditions for the production of levodione are pH values from4.0 to 9.0 and a temperature range of from 10 to 60° C. for a period of15 minutes to 72 hours.

More preferable conditions for the production of levodione are pH valuesfrom 5.0 to 8.0 and a temperature range of from 20 to 60° C. for aperiod of 30 minutes to 48 hours.

The concentration of ketoisophorone in a reaction mixture can varydepending on other reaction conditions, but, in general, is between 0.1g/l and 300 g/l, preferably between 1 g/l and 30 g/l.

Levodione produced enzymatically or biologically in a reaction mixtureas described above may be extracted by an organic solvent such as ethylacetate, n-hexane, toluene, or n-butyl. The extract may be analyzed byknown method such as gas chromatography, high performance liquidchromatography, thin layer chromatography or paper chromatography, orthe like. In case of gas chromatography, the following condition can beapplied as an example: Column: ULBON HR-20M (Shinwa, Japan) 0.25 mm×30m; Column temperature: 160° C. (constant); Injector temperature: 250°C.; Carrier gas: He (ca. 1 ml/min)

After the reaction, levodione in the reaction mixture may be recovered,for example, by extraction with a water-immiscible organic solvent,which readily solubilizes levodione, such as ethyl acetate, n-hexane,toluene, or n-butyl acetate. Further purification of levodione can beeffected by concentrating the extract to directly crystallize levodioneor by the combination of various kinds of chromatography, for example,thin layer chromatography, adsorption chromatography, ion-exchangechromatography, gel filtration chromatography or high performance liquidchromatography.

The following Examples further illustrate the present invention.

EXAMPLE 1 Partial Amino Acid Sequence of Enone Reductase of C. kefyr (C.macedoniensis) IFO 0960

The freeze-dried purified enone reductase (as described in EuropeanPatent Application No. 02003967.3 filed on Feb. 22, 2002) of C. kefyrwas digested with lysyl endopeptidase, and the resulting digest wasseparated by the Smart system, i.e., one nmol of the purified enzyme wasdissolved in 25 μl of 50 mM Tris-HCl buffer (pH 8.6) containing 8 Murea, and incubated at 37° C. for 1 hour. After this, 25 μl of 50 mMTris-HCl buffer (pH 8.6) were added to make the concentration of urea 4M. Then, 0.5 μl of 12 nmol/ml lysyl endopeptidase (Wako, Japan, 0.006nmol, E/S=1/167) was added, and incubated at 30° C. for 6 hours. Theresulting peptides were separated by the Smart system using thefollowing conditions: Column: μRPC C2/C18 SC2.1/10 (AmershamBioscience/Buckinghamshire, England); Flow rate: 100 μl/min; Liquid A:0.1% TFA; Liquid B: 0.1% TFA+80% CH₃CN; Gradient: 100% A (0-15 min);100% A→100% B (15-75 min); Column temperature: Room temperature;Detection: 214 nm, 280 nm.

The peptides (K-15, K-25.1, K-6.1, K-6.2, K30, K13.2, K-1.1, K-1.2,K-33, K-25.2, K-20, K-17, K-22, K-4.1, K-13.1, and K-9) were isolated,and the amino acid sequences of these peptides were analyzed with aprotein sequencer, i.e. by automated Edman degradation with a model491HT pulsed liquid protein sequencer (Applied Biosystems, Foster City,Calif.) to be Peptide K-15: SEQ ID NO: 3; PeptideK-25.1: SEQ ID NO: 4;Peptide K-6.1: SEQ ID NO: 5; Peptide K-6.2: SEQ ID NO: 6; Peptide K-30:SEQ ID NO: 7; Peptide K-13.2: SEQ ID NO: 8; Peptide K-1.1: SEQ ID NO: 9;Peptide K-1.2: SEQ ID NO: 10; Peptide K-33: SEQ ID NO: 11; PeptideK-25.2: SEQ ID NO: 12; Peptide K-20: SEQ ID NO: 13; Peptide K-17: SEQ IDNO: 14; Peptide K-22: SEQ ID NO: 15; Peptide K-4.1: SEQ ID NO: 16;Peptide K-13.1: SEQ ID NO: 17; and Peptide K-9: SEQ ID NO: 18.

The partial amino acid sequence obtained was compared with the sequencesof proteins stored in the SWISS-PROT (release 37.0+/06-14, June 99), PIR(release 60.0, March 99), and PRF (release 99-05, May 99) proteindatabases. Sequence alignment was performed by using Blast (J. Mol.Biol., 215, 403-410, 1990) and Fasta (Proc. Natl. Acad. Sci. USA, 85,2444-2448, 1988) programs. As a result, high homology with known OldYellow Enzymes was found.

EXAMPLE 2 Preparation of Chromosomal DNA of C. kefyr (C. macedoniensis)IFO 0960

The cells of C. kefyr (C. macedoniensis) IFO 0960 were cultivated in 200ml medium. Cells were collected by centrifugation and suspended in 10 mlTES buffer. 3 ml of 0.5 M EDTA, 0.5 ml of Zymolyase solution, and 0.5 mlof Proteinase K solution were added to the cell suspension. Afterincubation at 37° C. for 0.5 hour with gently mixing, 2 ml of 10% SDSwas added and mixed. After addition of H₂O to make the volume 20 ml, 10ml of TE-saturated phenol and 10 ml of chloroform were added and mixed.The upper layer was collected after centrifugation, the same volume ofphenol/chloroform was added and mixed. After centrifugation, upper layerwas collected and added with 0.1× volume of 3M sodium acetate and 2.5×volume of ethanol. Using a winding glass rod, DNA precipitate wascollected, rinsed with 70%, 80%, and 90% ethanol, dried and resuspendedin 5 ml of TE buffer containing 10 μl of 5 mg/ml RNase A. DNA wascompletely dissolved by gently mixing at 4° C. over night. 10 μl of 5mg/ml RNase A were added again, and the DNA solution was incubated at37° C. for 2 hours. After treatment with phenol/chloroform, water layerwas recovered and the DNA was ethanol precipitated, followed bycentrifugation. The pellet was resuspended in 50 ml of TE buffer.Concentration of thus obtained genomic DNA was 88 ng/μl.

EXAMPLE 3 Cloning of Partial Enone Reductase Gene of C. kefyr (C.macedoniensis) IFO 0960

Using the prepared genomic DNA as a template, a partial sequence for theenone reductase gene was obtained by degenerate PCR amplification usinga thermal cycler (Perkin-Elmer Cetus Instruments, USA). The degeneratePCR primers were designed based on the partial amino acid sequences(K-15, K-13.1, and K-9) obtained in Example 1, and were as follows:

Sense 1 (SEQ ID NO: 19)    GlyAspThrAsnIlePheLysProIle5′-GGIGATACIAATATATTTAAACCAAT-3′         C     C  T  C  G  T                 C        G                           C Anti 2 (SEQ IDNO: 20)    GlyGluLysThrPheThrTyrPheThr 5′-CCTTCTTTAGTAAAIGTATAAAAIGT-3′     C  C  T  G     G  G            G            C

The PCR reaction (50 μl) was carried out using 176 ng of chromosomal DNA(obtained in Example 2) as a template, 150 pmol each of degenerateprimer, 2.5 nmol each of dATP, dCTP, dGTP, and dTTP, 1.5 units of Ex Taqpolymerase (Takara Shuzo, Kyoto, Japan), and 5 μl of EX Taq buffer(Takara Shuzo). The initial template denaturation step consisted of 4min at 94° C. An amplification cycle of 1 min at 94° C., 1 min at 50°C., and 1.5 min at 72° C. was repeated for 35 times. After additional 10min reaction at 72° C., a DNA fragment containing a partial enonereductase gene (approx. 1 kb) was amplified. This fragment was cloned ona sequencing vector, and DNA sequence was determined by the dideoxychain-termination method. A Taq dye primer sequencing kit was used withan autosequencer (DNA Sequencer 373A, Applied Biosystems). The partialDNA sequence thus obtained for the enone reductase and deduced aminoacid sequence are as illustrated in SEQ ID NO:21 and SEQ ID NO:22,respectively.

EXAMPLE 4 Cloning of Complete Enone Reductase Gene of C. kefyr (C.niacedoniensis) IFO 0960

The inverse PCR was used to clone both upstream and downstream sequenceflanking the partial enone reductase DNA sequence obtained in Example 3.

1 μg of the genomic DNA of C. kefyr (C. macedoniensis) IFO 0960(obtained in Example 2) was digested with 10 units of Nco I (TakaraShuzo, Kyoto, Japan) in 50 μl of K-buffer containing 0.01% BSA. Afterovernight reaction at 37° C., the reaction mixture was treated withphenol/chloroform, the water layer was recovered and the DNA was ethanolprecipitated, followed by centrifugation. The DNA pellet was resuspendedin 1 ml of T4 DNA ligase buffer containing 700 units of T4 DNA ligase(Takara Shuzo). After overnight reaction at 15° C., the reaction mixturewas treated with phenol/chloroform, the water layer was recovered andthe DNA was ethanol precipitated, followed by centrifugation. The DNApellet was resuspended in TE buffer and used as a template for PCR. ThePCR primers were designed based on the partial enone reductase genesequence obtained in Example 3, and were as follows: IA1 (antisenseprimer for upstream region)=SEQ ID NO:23 and IS1 (sense primer fordownstream region)=SEQ ID NO: 24.

The PCR reaction (50 μl) was carried out using 250 ng of the templateDNA, 5 pmol each of primer, 2.5 nmol each of dATP, dCTP, dGTP, and dTTP,2.5 units of Ex Taq polymerase (Takara Shuzo), and 5 μl of EX Taq buffer(Takara Shuzo). The initial template denaturation step consisted of 4min at 94° C. An amplification cycle of 1 min at 94° C., 1 min at 60°C., and 4 min at 72° C. was repeated for 30 times. After additional 10min reaction at 72° C., a DNA fragment (approx. 4 kb) containing theupstream and downstream sequence of the enone reductase gene wasamplified. This fragment was cloned on a sequencing vector, and DNAsequence was determined.

By combining the thus obtained sequence with the partial enone reductaseDNA sequence obtained in Example 3, an estimated entire gene sequencecontaining its coding region as well as its regulatory region such as apromoter or a terminator was obtained. The estimated entire DNA sequencethus obtained for the enone reductase is illustrated in SEQ ID NO: 25containing the coding region as well as its flanking upstream anddownstream region (estimated ORF is 148-1359).

Next, the actual entire sequence of the enone reductase gene containingits coding region as well as its flanking upstream and downstream regionwas obtained by PCR as follows.

The genomic DNA of C. kefyr (C. macedoniensis) IFO 0960 (obtained inExample 2) was used as a template. The PCR primers were designed basedon the estimated enone reductase gene sequence obtained above (SEQ IDNO: 25), and are illustrated in SEQ ID NO: 26 (Sense) and SEQ ID NO: 27(Antisense).

The PCR reaction (50 μl) was carried out using 900 ng of the templateDNA, 10 pmol each of primer, 2.5 nmol each of dATP, dCTP, dGTP, anddTTP, 100 nmol of MgCl₂, 1.5 units of LA Taq polymerase (Takara Shuzo),and 5 μl of LA Taq buffer (Takara Shuzo). The initial templatedenaturation step consisted of 2 min at 94° C. An amplification cycle of1 min at 94° C., 1 min at 60° C., and 1.5 min at 74° C. was repeated for23 times. After additional 7 min reaction at 74° C., a DNA fragment(approx. 1.3 kb) containing the entire sequence of the enone reductasegene was amplified. This fragment was cloned on a sequencing vector, andthe DNA sequence was determined.

The entire DNA sequence thus obtained for the enone reductase containingits coding region as well as its flanking upstream and downstream regionis illustrated in SEQ ID NO:28 (ORF is 55-1266).

EXAMPLE 5 Expression of the Enone Reductase Gene and LevodioneProduction Using E. coli Having the Enone Reductase Gene of C. kefyr

A DNA fragment containing just the ORF of the enone reductase gene (1212bp) was obtained by PCR amplification. The PCR was performed withprimers, ExS (SEQ ID NO:29) and ExA (SEQ ID NO:30).

The vector carrying the entire sequence of the enone reductase gene(obtained in Example 4) was used as a template. The PCR reaction (50 μl)was carried out using 250 ng of the template DNA, 10 pmol each ofprimer, 2.5 nmol each of dATP, dCTP, dGTP, and dTTP, 1.5 units ofPyrobest DNA polymerase (Takara Shuzo), and 5 μl of Pyrobest buffer(Takara Shuzo). The initial template denaturation step consisted of 1min at 94° C. An amplification cycle of 0.5 min at 94° C., 1 min at 60°C., and 1.5 min at 75° C. was repeated for 15 times. After additional 5min reaction at 75° C., a DNA fragment (approx. 1.2 kb) containing justthe ORF of enone reductase gene was amplified.

This amplified fragment of the enone reductase gene was cloned on avector, pET101/D-TOPO, using a pET Directional TOPO® Expression Kits(Invitrogen Corporation, USA) according to an instruction manualprepared by the manufacturer. The vector carrying the enone reductasegene thus obtained (pET101/D-TOPO-ER) was introduced into E. coli BL21(DE3), and several clones were selected for sequence analysis using anautomatic sequence analyzer (DNA Sequencer 373A, Applied Biosystems).One of the clones, E. coli BL21 (DE3)[pET101/D-TOPO-ER], that showedcompletely the same sequence as the enone reductase sequence of C. kefyrwas selected for further experiments. The strain, E. coli BL21(DE3)[pET101/D-TOPO] was also prepared as a control.

Each of the strains, E. coli BL21 (DE3)[pET101/D-TOPO-ER] and E. coliBL21 (DE3)[pET101/D-TOPO], was inoculated into the M9 minimum medium (5ml in tube) containing 0.05 mg/ml of ampicillin and 2% (W/V) of casaminoacids (Difco laboratories, USA) and cultivated at 37° C. When theoptical density at 610 nm reached 0.4, IPTG (isopropylbeta-D-thiogalactopyranoside) was added to the medium to make theconcentration 0.01 mM and cultivation was continued for further 8-10hours. Then the cells were collected by centrifugation, and a portion ofthe cells was used for SDS-PAGE analysis. As a result, an IPTG-inducedprotein band estimated as 45 kDa was observed only when the recombinantstrain, E. coli BL21 (DE3)[pET101/D-TOPO-ER] was used.

The rest of the collected cells was resuspended into 2 ml of 100 mMpotassium phosphate buffer (pH 7.0). The suspension was used forconfirming an activity to produce levodione from ketoisophorone. Thissuspension was divided into two portions (1 ml each), and the reactionwas started by adding 33 mM (final concentration, hereinafterabbreviated as f.c.) of ketoisophorone and 280 mM (f.c.) of D-glucosewith or without 0.37 mM (f.c.) of NAD⁺, 15 units/ml (f.c.) of glucosedehydrogenase. The reaction was carried out at 30° C. overnight. Thereaction mixture was extracted with ethylacetate to recover levodione inthe ethylacetate layer. The extract was analyzed by gas chromatography.As a result, levodione was detected only when the recombinant strain, E.coli BL2 1 (DE3)[pET101/D-TOPO-ER] was used.

1. A process for the production of levodione, which comprises contactingketoisophorone with an enzyme derived from Candida or Zygosaccharomyceswhich has enone reductase activity, wherein the enzyme is characterizedby the following physico-chemical properties: (a) molecular mass:61,300±5,000 Da as determined by gel filtration; (b) co-factor: NADPHand NADH; (c) substrate specificity: active on α,β-unsaturated ketones;(d) optimum temperature: 55-60° C. at pH 7.4; and (e) optimum pH: pH4.5-8.5, in the presence of NADPH or NADH and isolating the resultinglevodione from the reaction mixture.
 2. The process of claim 1, whereinthe ketoisophorone is contacted with the enzyme at pH values in therange of from 5.0 to 8.0 and at a temperature in the range of from 20 to60 ° C. for 15 minutes to 48 hours.
 3. The process of claim 1, whereinthe enzyme is derived from Candida.
 4. The process of claim 3, whereinthe enzyme derived from Candida is derived from Candida kefyr.
 5. Theprocess of claim 4, wherein the enzyme derived from Candida kefyr isderived from Candida kefyr IFO
 0960. 6. The process according to claim1, wherein the enzyme is a polypeptide having the amino acid sequenceshown in SEQ ID NO: 2 or is encoded by a polynucleotide that is at least90% identical to a polynucleotide that encodes the polypeptide havingthe amino acid sequence shown in SEQ ID NO: 2 and has enone reductaseactivity.
 7. The process according to claim 1, wherein the enzyme is apolypeptide having the amino acid sequence shown in SEQ ID NO:
 2. 8. Aprocess of claim 1, wherein ketoisophorone is contacted with the enzymeat pH values in the range of from 4.5 to 8.5 and at a temperature in therange of 10 to 60° C. for 5 minutes to 72 hours.